Segmental bone defects resulting from delayed or non-union of fractures, congenital defects, or resection of bone metastases are a significant clinical concern and cost the U.S. healthcare system greater than $28 billion per year. Delivery of growth factors such as bone morphogenetic protein 2 (BMP-2) from collagen sponges is an effective clinical alternative to autografts and allografts in the treatment of bone defects and shows the economic viability of these approaches, with sales of nearly $1 billion for Medtronic's Infuse system. However, safety concerns such as ectopic bone growth due to rapid and uncontrolled release of BMP-2 limit FDA approved applications of this delivery system to lumbar spinal fusion and tibial fracture. We propose to develop a hydrogel delivery system based on a family of proteins called keratins that will serve as a matrix for infiltrating regenerative cells nd as a means to tailor BMP-2 delivery. The inherent disulfide cross-links found in keratin can be modulated by simple chemical modification, thereby controlling the rate of scaffold degradation. Because BMP-2 delivery correlates with scaffold degradation, modulation of the disulfide cross-linking provides a means to tailor the delivery of BMP-2 for safe and effective bone healing. The long-term goal of this research is to develop a biomaterial superior to existing systems by tailoring the delivery of growth factors such as BMP-2 for optimal healing response within the context of a material that can provide a physical matrix to regenerating bone cells. The specific goal of this proposal is to demonstrate the effect of modulating disulfide cross-linking in keratin hydrogels on rates of scaffold degradation, BMP-2 release, and BMP-2 bioactivity to promote bone regeneration. We will use three aims to achieve this goal: Aim 1: Modulate the disulfide cross-linking density inherent in keratin hydrogels to promote favorable material and biological properties. Aim 2: Determine release kinetics and bioactivity of BMP-2 delivered from keratin hydrogels. Aim 3: Assess the effect of modulating the rate of BMP-2 delivery with keratin hydrogels on bone regeneration in a critically-sized segmental rat femur defect model. The proposed experiments will specifically address the ability of keratin hydrogels to promote improved bone regeneration and safety profiles compared to existing biomaterial carriers. More generally, these studies will demonstrate the utility of keratin hydrogels as a tunable delivery platform for growth factors in restorative medicine approaches that seek to use therapeutic molecules to direct healing in states of injury or disease.